The 19-transmembrane multisubunit γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimer's disease (AD) by intramembrane proteolysis of the β-amyloid precursor protein (APP). Despite substantial advances in elucidating how this protein complex functions, the effect of the local membrane lipid microenvironment on γ-secretase cleavage of substrates is still poorly understood. Using detergent-free proteoliposomes to reconstitute purified human γ-secretase, we examined the effects of fatty acyl (FA) chain length, saturation and double-bond isomerization, and membrane lipid polar headgroups on γ-secretase function. We analyzed γ-secretase activity and processivity [i.e., sequential cleavages in the APP transmembrane domain that convert longer Aβ species (e.g., Aβ 46 ) into shorter ones (e.g., Aβ 40 )] by quantifying the APP intracellular domain (AICD) and various Aβ peptides, including via a bicine/urea gel system that detects multiple Aβ lengths. These assays revealed several trends. (1) Switching from a cis to a trans isomer of a monounsaturated FA chain in phosphatidylcholine (PC) increased γactivity, did not affect Aβ 42 :Aβ 40 ratios, but decreased the ratio of long (≥42) versus short (≤41) Aβ peptides. ( 2) Increasing the FA carbon chain length (14, 16, 18, and 20) increased γ-activity, reduced longer Aβ species, and reduced the Aβ 42 :Aβ 40 ratio. (3) Shifting the position of the double bond in 18:1(Δ9-cis) PC to the Δ6 position substantially reduced activity. (4) Gangliosides increased γ-activity but decreased processivity, thus elevating the Aβ 42 :Aβ 40 ratio. (5) Phosphatidylserine decreased γ-activity but increased processivity. ( 6) Phosphatidylinositol strongly inhibited γ-activity. Overall, our results show that subtle changes in membrane lipid composition can greatly influence γ-secretase activity and processivity, suggesting that relatively small changes in lipid membrane composition may affect the risk of AD at least as much as presenilin or APP mutations do.
The 19-transmembrane γ-secretase complex generates the amyloid β-peptide of Alzheimer’s disease by intramembrane proteolysis of the β-amyloid precursor protein. This complex is comprised of presenilin, Aph1, nicastrin, and Pen-2. The exact function and mechanism of the highly conserved Pen-2 subunit remain poorly understood. Using systematic mutagenesis, we confirm and extend our understanding of which key regions and specific residues play roles in various aspects of γ-secretase function, including maturation, localization, and activity, but not processivity. In general, mutations (1) within the first half of transmembrane domain (TMD) 1 of Pen-2 decreased PS1 endoproteolysis and γ-secretase proteolytic activity, (2) within the second half of TMD1 increased proteolytic activity, (3) within the cytosolic loop region decreased proteolytic activity, (4) within TMD2 decreased PS1 endoproteolysis, (5) within the first half of TMD2 decreased proteolytic activity, and (6) within C-terminal residues decreased proteolytic activity. Specific mutational effects included N33A in TMD1 causing an increase in γ-secretase complexes at the cell surface and a modest decrease in stability and the previously unreported I53A mutation in the loop region reducing stability 10-fold and proteolytic activity by half. In addition, we confirm that minor PS1 endoproteolysis can occur in the complete absence of Pen-2. Together, these data suggest that rather than solely being a catalyst for γ-secretase endoproteolysis, Pen-2 may also stabilize the complex prior to PS1 endoproteolysis, allowing time for full assembly and proper trafficking.
The 19-transmembrane, multi-subunit γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β-amyloid precursor protein (APP). The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen-2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high-resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen-2 can be purified from bacteria to >95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N-terminal MBP tag on Pen-2 still permits incorporation into the complex and subsequent presenilin-1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen-2 and the γ-secretase complex.
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